Lack of presence of the human cytomegalovirus in human glioblastoma

Abstract

Recent reports have indicated human cytomegalovirus (HCMV) to be associated with human glioblastoma carcinogenesis. In established examples of viral carcinogenesis, viral DNA and one or more of its products have been detected in most tumor cells of biopsies in the majority of cases. To test whether HCMV is associated with human glioblastoma based on this criterion, we measured the number of viral DNA molecules per cell in both frozen and paraffin-embedded tumor biopsies from 58 patients using real-time quantitative PCR (QPCR). Immunohistochemical and fluorescence in situ hybridization (FISH) to detect HCMV proteins and genome was performed in 10 cases using formalin-fixed paraffin-embedded glioblastoma tissues. Southern blotting using DNA extracted from four glioblastoma cell lines together with immunoblotting using the four cell lines and five glioblastoma tissue samples were also performed. We further confirmed the immunoblot bands using liquid chromatography-tandem mass spectrometry assay. As a result, HCMV DNA was not detected in the tumor cells from any of the glioblastoma cases by QPCR detecting two different HCMV genes, in clear contrast to samples from patients with HCMV infection. Southern blotting and immunoblotting of cell lines and FISH using paraffin sections were all negative. However, immunoblotting and immunohistochemistry using tissue samples were partly positive, but HCMV proteins were not detected by proteomic analysis, suggesting false positivity of the analyses. As our QPCR analysis could detect 10 copies of HCMV DNA mixed with DNA extracted from 10 4 HCMV-negative cells, we conclude that HCMV is not persistent, at least in the tumor cells, of developed human glioblastoma.

The association between human cytomegalovirus and glioblastomas: a review

“…Interestingly, the authors noted that the HCMV positivity demonstrated in a few samples were actually the HCMV antibodies binding to non-viral human proteins such as human albumin and myelin basic protein. [34] This finding suggests a previously unknown cross-reactivity among HCMV antibodies. Another study by Holdhoff et al [35] assessed whether altering experimental conditions could generate false positive results.…”

“…[61] A few recent studies may shed light onto why there is such discrepancy observed in the literature. A study by Yamashita et al [34] attempted to detect HCMV in GBM samples through a wide range of detection methods. They were unable to detect the presence of HCMV in many of the samples.…”

“…The last hypothesis is that these observations are merely an experimental artifact. Several recent publications have outlined possible scenarios where detection of HCMV may be due to experimental artifact including cross-reactivity of antibodies, [34] the concentration of antibodies, [35] or presence of expression vector genetic material in sequencing datasets. [36] …”

Identification of a Natural Killer Cell Receptor Allele That Prolongs Survival of Cytomegalovirus-Positive Glioblastoma Patients

“…Of our entire glioblastoma patients, 30.5% were pp65 positive in tumor, whereas 12% had CMV DNA in blood. The latter may be indicative of reactivation as a consequence of changes in the immune function of patients as CMV generally establishes latency after primary infection during childhood or adolescence (55). One difficulty is that there is currently no consensus regarding the best method for demonstrating CMV positivity in glioblastoma patients.…”

1410 Absence of human cytomegalovirus infection in childhood brain tumors

“…For this study we used the automated method and the commercially available antibody (prediluted antibody cocktail, clone DDG9/CCH2, reacting with an immediate early antigen and an early CMV antigen, DDG9 reacts with 76 kD protein, whereas CCH2 reacts with a 43 kD protein) that the community routinely use for diagnostic purpose on formalin fixed and paraffin embedded specimens. This choice was based on the fact that the methodological changes that improve the sensibility of the immunoreactions also might potentially increase the risk of false positive as recently demonstrated [11]. On the other hand it has been suggested that the standard immunohistochemical protocols might not recognize a low viral load.…”